Movement of free fatty acids (FFA) between intra and extra-cellular sites is an essential part of the mammalian energy cycle. At many points in this cycle, FFA must cross a cellular membrane. Understanding the molecular mechanisms governing this movement is important since: 1) FFA are quantitatively the most important physiological energy source; 2) FFA are potent modulators of cellular behavior; and 3) this cycle provides a paradigm for the movement of lipophilic molecules, in general. Defects in any of these steps in the cycle may result in nutritional imbalances and/or various pathologies associated with altered levels of FFA. Because of the physiological importance of FFA, as well as the potential implication for amphiphile transport, in general, the FFA transport problem has received considerable attention. There is, however, no consensus: even in the most recent literature, as to whether the plasma membrane is rate limiting for FFA transport. A significant number of recent studies indicate that plasma membrane proteins play a key role as transport proteins in the cellular uptake of long chain FFA. Virtually all studies in model membranes, however, have demonstrated that FAA can spontaneously transfer through the bilayer of pure lipid vesicles, raising the question of why transport through the lipid phase of the plasma membrane does not short circuit the protein mediated transport. In fact, because of this observation, it has generally been argued that FFA (and, in fact, most lipohile transport) is not protein mediated. On the other hand, our own studies, while agreeing that FFA do spontaneously transfer through the lipid bilayer, show that for long chain fluorescence FFA the rate of flip-flop is much slower than the off rate, contrary to conventional views of amphipath permeability through lipid bilayers. In preliminary studies, we have found that the barrier to flip-flop is a sensitive function of membrane lipid composition, physical state, and bilayer size, which raises the possibility that lipid factors may slow flip-flop below the rate necessary for physiological function in at least some biological membranes. In the proposed research, we will investigate the flip-flop mechanism of both fluorescent and natural FFA in lipid vesicles, in vesicles formed from red cell and adipocyte plasma membranes, and in lipid vesicles reconstituted with a FFA transport protein. Our methods of analysis and interpretation will be based upon the rigorous use of a specific kinetic model. Various experimental and theoretical techniques will be developed to establish the validity of this model. With this model as basis, we will then carry out studies to determine how lipid composition and FFA structure and charge state affect the intrinsic rate constants of the model. While the experimental techniques needed to carry out these studies with fluorescent FFA represent a straightforward extension of our previous work, new methods will be developed for similar studies of natural FFA.